Purification and Characterization of Glucosamine Oxidase from Aspergillus Terreus NUK-1204 for Production of Glucosaminic Acid

Research Article

Austin J Biotechnol Bioeng. 2023; 10(1): 1121.

Purification and Characterization of Glucosamine Oxidase from Aspergillus Terreus NUK-1204 for Production of Glucosaminic Acid

Shuen-Fuh Lin*; Pang-Hung Huang

Department of Life Sciences, National University of Kaohsiung, 700, Kaohsiung University Road, Taiwan

*Corresponding author: Shuen-Fuh Lin Department of Life Sciences, National University of Kaohsiung, 700, Kaohsiung University Road, Nanzih District, Kaohsiung, 811, Taiwan. Tel: +886 7 5919227; Fax: +886 7 5919404 Email: sflin@nuk.edu.tw

Received: August 21, 2023 Accepted: September 16, 2023 Published: September 23, 2023

Abstract

D-Glucosaminic acid (2-amino-2-deoxy-D-gluconic acid) is a valuable glucosamine derivative from the oxidation of the glucosamine (2-amino-2-deoxy-D-glucose). It can be produced via the fermentation of certain microorganisms, which involves complicated purification task, though. In an attempt to find an enzyme suited to glucosaminic acid production, the study presented a purified homogeneous glucosamine oxidase deriving from the wheat bran culture of a soil-isolated fungal strain, Aspergillus terreus NUK-1204. Maximum activity was observed in the wheat bran solid culture after five days of NUK-1204 growth, following its release from cells at 0.011 unitmL -1culture filtrate. The new kind of oxidase comprises a single polypeptide chain with a molecular mass of 54 kDa, containing FAD and exhibiting absorption maxima at 274, 372 and 439 nm. The enzyme was stable in 4.5-11 pH range, with an optimal reaction pH at 10.5 and optimal reaction temperature at 30°C. It remained stable at 20-50°C for 1 hr. The relative activity of glucosamine oxidase towards glucosamine and N-acetyl-glucosamine was 100:7, demonstrating high substrate specificity. It doesn’t inhibit glucosamine. To the best of our knowledge, this is the first report ever on the discovery of a glucosamine oxidase based on enzyme substrate specificity.

Keywords: Glucosamine oxidase; Aspergillus terreus; Glucosaminic acid

Introduction

There have been studies on the biosynthesis of D-Glucosaminic acid (2-amino-2-deoxy-D-gluconic acid), a component of bacterial lipopolysaccharides with species of a-amino acid, most involving the oxidation of D-glucosamine (2-amino-2-deoxy-D-glucose) via a specific membrane oxidase in the bacteria [1]. While knowledge on this enzyme is still limited, D-glucosaminic acid has been used as sweetener, condiments, and chiral synthon [2]. It also belongs to a chiral pool and is a trigger for the asymmetric synthesis of various amino acids, such as 4,5,6-trihydroxynorleucine [3], and several glycosidase inhibitors, like (+)-castanosporspermine [4], plus an expanding scope of potential applications. Carbohydrate oxidation has been widely applied in pharmaceutical production as biocatalyst and diagnostic reagent. Given the key role of regioselectivity and steroselectivity in these reactions, it is critical to find optimal biocatalysts [5,6]. Various sugar oxidoreductases have been identified from a variety of sources, including bacteria [7], fungi [8,9], and algae [10,11], most of which being active with mono and disaccharides primarily, with only a few exhibiting high specificity toward amino-sugar. One example is N-acyl-D-hexosamine oxidase obtained from Pseudomonas sp. [12], which shows high reactivity to N-acyl-D-hexosamines containing N-acyl-D-glucosamine and N-acyl-D-galactosamine. In contrast, D-glucosamine and D-glucose demonstrated lower activity to the enzyme.

In line with the proposal of Danneel et al. [13], the study applied a rapid and convenient screening method for the in situ assay of sugar oxidase-producing soil fungi, on top of under solid-state fermentation and the peroxidase-chromogen method for assaying oxidase activity. Strain NUK-21, identified as Myrmecridium flexuosum, was isolated and named lactose oxidase, based on its substrate specificity [14]. Fortunately, strain NUK-1204, identified as Aspergillus terreus, was isolated. Oxidase deriving from the purification of the aqueous extract of wheat bran culture of this strain showed high reactivity toward D-glucosamine, while D-N-acetylglucosamine and D-glucose exhibited lower activity. The new kind of oxidase was named glucosamine oxidase, based on its substrate specificity. Its purification and some properties follow.

Materials and Methods

Chemicals

Fractogel DEAE-650M, HW-55 gel filtration resins were purchased from Merck (Darmstadt, Germany). Toyopearl Phenyl-650M was purchased from Toyo Soda Manufacturing (Tokyo, Japan). Ultragel-HA was acquired from IBF Biotechnics (Pairs, France). Protein assay dye and SDS-PAGE molecular weight standards were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Sephacryl S-200 HR16/60 FPLC column was from GE Healthcare Bio-Science (Uppsala, Sweden). FPLC molecular weight standards, 4-aminoantipyrine (4AA), peroxidase and all the sugars were from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytic reagent grade.

Microorganisms and Culture Conditions

The fungal strain NUK-1204 was isolated from the soil, identified as Aspergillus terreus and deposited at the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) of Taiwan under accession number BCRC 930228. The organism was maintained on YM agar slants (1% glucose, 0.5% peptone, 0.3% malt extract, and 0.3% yeast extract) at 30°C.

Production of Glucosamine Oxidase

The NUK-1204 strain produced glucosamine oxidase activity on wheat bran culture. For enzyme production, this strain was taken from a slant culture and inoculated in 250 ml flasks, each of which contained 5g of wheat bran supplemented with 17.5 ml of water. After 5 days of cultivation at 30°C, the entire culture was used for enzyme preparation.

Enzyme Assay

The enzyme activity was assayed through the peroxidase-4AA method. An aliquot of enzyme was incubated at 30°C in 1 mL of 50 mM Tris–HCl buffer (pH 7.8, buffer A) containing 50 mM glucosamine, 1 U peroxidase, 0.1 mM 4AA, and 1 mM phenol. The increase in optical density was measured at 500 nm for 1 min. The enzyme activity was estimated by monitoring the consumption of oxygen with an oxygraph (Oxy-5, Gilson Medical Electronic, France). The reaction was initiated by the addition of appropriate amounts of the enzyme to the reaction mixture containing 25 mM glucosamine in buffer A, and the initial velocity of oxygen consumption was measured. One unit of enzyme activity was defined as the amount of enzyme that produced 1 μmolmin-1 of H2O2 or consumed 1 μmol min-1 of O2 at 30°C.

Enzyme Purification

A typical purification scheme of the glucosamine oxidase from wheat bran culture is described as follows. All operations were conducted at 4°C.

Preparation of Crude Extract

The wheat bran culture of strain NUK-1204 (120g) was soaked in 1 l of 20 mM borate buffer (pH 9.5) with 0.1% triton X-100 for 30 min and squeezed through a fine-mesh cloth. The aqueous extract was then centrifuged at 8000g for 30 min to remove particles.

Ammonium Sulfate Fractionation

The clarified aqueous extract (830 mL) was brought to 65% saturation with ammonium sulfate; the precipitate was removed via centrifugation at 9,000×g at 4°C for 30 min. The resulting filtrate was collected via centrifugation.

Toyopearl Phenyl-650M Column Chromatography

The 65% saturated ammonium sulfate filtrate was applied to a Toyopearl phenyl-650M column (2.5×30 cm) (Toyo Soda Manufacturing), which was pre-equilibrated with buffer A containing 65% saturated ammonium sulfate. Glucosamine oxidase was eluted with a 1,400 mL linear gradient of 65–0% ammonium sulfate in buffer A at a flow rate of 2.5 ml/min.

Fractogel HW-55 Column Chromatography

The enzyme solution was applied to a Fractogel HW-55 column (2.5cm×115cm), which was pre-equilibrated with 50 mM buffer A at a flow rate of 0.25 ml/min. To concentrate the pooled active fractions (352 mL), ammonium sulfate was added to the enzyme solution to a final concentration of 2M and then recharged to a small Toyopearl phenyl-650M column (1cm×6 cm), which was pre-equilibrated with buffer A containing 2M ammonium sulfate. In this process, the enzyme was eluted directly with buffer A. The volume of pooled active fractions was 15 mL.

Fractogel DMAE-650 Column Chromatography

The enzyme solution (70 ml) was applied to a Fractogel DMAE-650 column (2.5cm×30 cm), which was pre-equilibrated with buffer A. The enzyme was eluted by linear gradient from 0 to 0.15 M NaCl in buffer A at a flow rate of 2.0 ml/min.

Ultragel-HA Hydroxylapatite Column Chromatography

The pooled active fractions were further purified by applying them to an Ultragel-HA hydroxylapatite column (2.5cm×15 cm), which was pre-equilibrated with 10 mM phosphate buffer (pH 7.0). The enzyme was eluted with a 600-mL linear gradient of 10–150 mM phosphate buffer at a flow rate of 1.5 ml/min. The active fractions were pooled and stored at -20°C.

Amino-Terminal Amino Acid Sequence of Glucosamine Oxidase

Amino acid sequence of the purified peptide (0.01mg) was determined by an automated Edman degradation with an Applied Biosystems, Procise 494 protein sequencer (Foster City, CA, USA).

Other Analytical Methods

The molecular mass of the native enzyme was estimated through gel filtration under the following conditions: system, Fast Performance Liquid Chromatography (FPLC) system; pump, P-500; controller, LCC-500; detection, absorbance at 280 nm; column, Sephacryl S-200 HR16/60 FPLC column; solvent, 100 mM NaCl in 10 mM acetate buffer (pH 5.5). The molecular mass of the denatured enzyme was determined through SDS polyacrylamide gel electrophoresis (SDS-PAGE) on 10% acrylamide slabs using a modified Laemmli buffer system [15]. Protein concentrations were determined through the Bradford method with bovine serum albumin as the standard [16]. Protein assay reagents were purchased from Bio-Rad. The Mass spectrometry data were obtained using APCI ion source from Advion expression® CMS instrument (Advion Inc., Ithaca, NY, USA). Nuclear magnetic resonance spectra were recorded on a Varian-Mercury plus 300 MHz spectrometry (Agilent, Santa Clara, CA, USA). 300 MHz for 1H and 75 MHz for 13C with D2O (deuterium oxide) as the solvent.

Thin-Layer Chromatography (TLC) Analysis of the Reaction Product

A reaction mixture (1mL) containing 2.5% glucosamine (w/v), 2 units of glucosamine oxidase, and 10 units of catalase was incubated at 30°C with a reciprocal shaker (120 rpm). Subsequently, the reaction product was detected using Thin-Layer Chromatography (TLC) with silica gel 60 plates (Merck, Germany). Aliquots (approximately 1μL) of sample were applied to the TLC plate and developed in n-butanol/methanol/ammonium water/water (5:4:2:1, v/v). The plate was sprayed with 50% sulfuric acid dissolved in ethanol /water (1:1 v/v) and then heated at 150°C until charring occurred.

Results

Enzyme Production

Extracellular glucosamine oxidase deriving from Aspergillus terreus NUK-1204 was produced in Solid-State Fermentation (SSF) only, rather than submerged culture. Compared with rice bran, corn flour, sugarcane bagasse, and soy meal, what bran was found to be the best medium for enzyme production, as with application of wheat bran as a substrate in SSF, the addition of other carbon sources (e.g., glucose, fructose, glycerol, starch, and cellulose) or nitrogen sources (e.g., peptone, yeast extract, malt extract, and urea) didn't enhance enzyme production. A mixture of wheat bran and water in the ration of 1:3.5 (w/v) was found to be the optimal fermentation medium. In enzyme product, it typically took five days at 30o to reach maximum glucosamine oxidase activity (Figure 1). About 0.011 U of enzyme activity per mL could be extracted from the culture broth with 20 mM borate buffer (pH 9.5) containing 0.1% triton X-100.